Manganese is an essential trace nutrient that is also potentially toxic. In mammals, manganese is a potent neurotoxin and has been implicated in Parkinson's disease-like syndromes. Yet surprisingly little is known regarding the cell biology of this metal and its mechanisms of toxicity. We have been exploiting the bakers' yeast S. cerevisiae as a model system to characterize eukaryotic factors that control manganese homeostasis. Over the past funding period, we have identified a number of such factors including: Smf2p, a NRAMP metal transporter that localizes to intracellular vesicles and plays a central role in manganese trafficking; Mtm1p, a putative manganese transporter for the mitochondria; and phosphate metabolism factors that are critical determinants of manganese toxicity. Through four specific aims, we will continue our investigations of these and other manganese homeostasis factors. Our goals are to (1) Define the nature of the Smf2p manganese transport vesicles and to address the role of endocytosis in the uptake and intracellular trafficking of manganese; (2) Elucidate the role of yeast and human Mtm1p in the transport of manganese into the mitochondrial matrix; (3) Determine how phosphate metabolism pathways modulate cellular resistance to manganese toxicity; and (4) Employ yeast genetics to identify new manganese homeostasis factors, including putative manganese metallochaperones. Overall, these studies will combine diverse disciplines of yeast molecular genetics, biochemistry and cell biology to provide new insight into the homeostasis of essential, but potentially toxic manganese ions.